Abstract

Very long chain aliphatic compounds occur in the suberin polymer and associated wax. Up to now only few genes involved in suberin biosynthesis have been identified. This is a report on the isolation of a potato (Solanum tuberosum) 3-ketoacyl-CoA synthase (KCS) gene and the study of its molecular and physiological relevance by means of a reverse genetic approach. This gene, called StKCS6, was stably silenced by RNA interference (RNAi) in potato. Analysis of the chemical composition of silenced potato tuber periderms indicated that StKCS6 down-regulation has a significant and fairly specific effect on the chain length distribution of very long-chain fatty acids (VLCFAs) and derivatives, occurring in the suberin polymer and peridermal wax. All compounds with chain lengths of C(28) and higher were significantly reduced in silenced periderms, whereas compounds with chain lengths of C(26) and lower accumulated. Thus, StKCS6 is preferentially involved in the formation of suberin and wax lipidic monomers with chain lengths of C(28) and higher. As a result, peridermal transpiration of the silenced lines was about 1.5-times higher than that of the wild type. Our results convincingly show that StKCS6 is involved in both suberin and wax biosynthesis and that a reduction of the monomeric carbon chain lengths leads to increased rates of peridermal transpiration.

StKCS6 transcript profile in potato organs and tuber tissues and its down-regulation in StKCS6-silenced lines. (A) Relative StKCS6 transcript accumulation (log-transformed) in potato stem (S), leaf (L), root (R), tuber-parenchyma (T-PAR), and tuber periderm (T-PER) was determined by real-time RT-PCR analysis. Values were normalized using the housekeeping reference gene adenine phosphoribosyl transferase. The data represent the mean ±sd of three replicates. (B) RT-PCR analysis with PCR incremental cycle numbers of the StKCS6 gene, to verify silencing of the StKCS6 transcript. Samples correspond to potato tuber periderms of independent StKCS6-RNAi transgenic lines. PCR products were analysed at the cycle numbers indicated in the top. Equal amount of cDNA was used as template for each sample as showed by the PCR amplification of StACTIN. Note that lines 5, 9, and 34 showed the highest StKCS6 down-regulation whereas line 23 was partially silenced and line 37 was non-silenced, showing an accumulation of the StKCS6 transcript comparable to that of the wild type.

Total amount of suberin and wax compounds per surface area (μg cm−2). The soluble fraction (wax) was obtained by treating the enzymatically isolated periderms with a mixture of chloroform:methanol. The suberin fraction was obtained after trans-esterification of wax-free periderms and the released monomers were classified as suberin aromatic or aliphatic compounds. Note that as a whole, periderms from 21-d-stored tubers showed an increased load of suberin and wax. Data represent the mean ±sd of the periderms from StKCS6-RNAi (suberin n=8: line 5 n=3, line 9 n=2, line 34 n=3; wax n=7: line 5 n=3, line 9 n=2, line 34 n=2) and wild-type (WT) (suberin n=5; wax n=7) tubers.

Chemical composition of suberin and wax fractions of StKCS6-RNAi and wild-type periderms from 21-d-stored tubers. (A) Absolute amounts (μg cm−2) of suberin monomers released after trans-esterification of wax-free periderms. (B) Absolute amounts (μg cm−2) of wax compounds obtained by treating the enzymatically isolated periderms with a mixture of chloroform:methanol. StKCS6-RNAi periderms show a decrease in C28 and longer VLCFA and derivatives of all substance classes, both in suberin and wax. Data represent the mean ±sd of the periderms from StKCS6-RNAi (suberin n=8: line 5 n=3, line 9 n=2, line 34 n=3; wax n=7: line 5 n=3, line 9 n=2, line 34 n=2) and wild-type (suberin n = 5; wax n=7) tubers.

Periderm water permeances of StKCS6-RNAi and wild-type tubers from freshly-harvested and 21-d-stored tubers. After 21 d storage, periderms improve the water barrier properties showing a decreased water permeance (P; m s−1). At this stage, the StKCS6-silenced periderms have a 1.54-fold increase on water permeance when compared to wild-type (t test; **, P < 0.01). Data represent the mean and error bars the 95% confidence intervals for the periderms from wild-type freshly-harvested (n=11) and 21-d-stored (n=14) and for the StKCS6-RNAi freshly-harvested (line 5 n=7; line 34 n=11) and 21-d-stored (line 5 n=9; line 34 n=11) tubers.

Ultrastructure of StKCS6-silenced periderms. Transmission electron micrographs showing a detailed view of the cork cell walls ultrastructure of wild-type (A) and transgenic StKCS6-RNAi (B) periderms. The polysaccharide primary wall (PW) and tertiary wall (TW) as well as the suberized secondary wall (SW) formed by the typical suberin lamella show a normal development in the StKCS6-RNAi periderm.